Combination stirrer

ABSTRACT

The present patent application describes a stirrer comprising a combination of at least one axially-conveying element and at least one radially-conveying element relative to the rotary shaft of the stirrer wherein the largest diameter of the at least one axially-conveying element is equal to or less than the inner diameter d i  of the radially-conveying element. In one embodiment the stirrer according to the invention is a combination of one anchor stirrer with at least one inclined-blade stirrer. Furthermore the use of the stirrer according to the invention for the culture of cells in a dialysis method is described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.13/375,153, internationally filed Jun. 2, 2010, issued as U.S. Pat. No.10,076,731 on Sep. 18, 2018, which is a National Stage Application under35 U.S.C. § 371 of International Application No. PCT/EP2010/003356,filed Jun. 2, 2010, which claims priority to and the benefit of EuropeanPatent Application No. 09007457.6, filed Jun. 5, 2009, the entiredisclosures of each of which are incorporated herein by reference intheir entirety.

Herein is reported an agitator, a device comprising the agitator and theuse of the agitator for the cultivation of cells. The agitator comprisesat least one axially-conveying element and at least oneradially-conveying element, such as the combination of an anchorimpeller as a radially-conveying element and one or more inclined-bladeimpeller(s) as an axially-conveying element. The use of the agitatorresults in an improved mixing and in particular a lower biofouling indialysis processes as well as a higher mass transfer rate.

TECHNOLOGICAL BACKGROUND

The production of recombinant proteins, vaccines and antibodies with theaid of prokaryotic and eukaryotic cells plays an essential role inmodern pharmaceutical production. To produce complexpost-translationally modified proteins and antibodies animal derivedcells are primarily used. However, the use of animal derived cells setshigh demands for the fermentation process due to the specificcharacteristics of these cells such as e.g. the culture medium, thesensitivity towards limitation and inhibitions (for example by lactate,CO₂, ammonium etc.), the sensitive outer membrane (shear stress), thelow specific rates and the sensitivity towards variations in the cultureconditions (e.g. due to local inhomogeneities, pH variations, pO₂variations etc.). These properties have to be taken into considerationwhen designing bioreactors and for process control.

In recent years various types of reactors for culturing cells have beendeveloped. Irrespective of the type, the reactor must be able to fulfillthe following basic technical functions: adequate suspension as well ashomogenization, adequate material and heat transport as well as aminimal shear stress on the cells. The stirred-tank reactor isespecially suitable for industrial use. In this reactor the necessaryenergy for fulfilling the basic functions is introduced by mechanicalstirring.

In order to achieve high product titer and specification compliantproduct quality, the operating mode of the reactor in particular playsan important role in addition to cell line development, mediacomposition and design of the reactor. Generally the following operatingmodes are employed: batch processes, fed batch processes, continuousprocesses with or without cell retention (for example perfusion orchemostat) as well as semi-continuous processes such as e.g. internal orexternal dialysis.

To prevent nutrient limitations feed solutions are added to reactor inthe form of concentrated solutions (so-called fed batch processes). Toavoid inhibition by end products of the cell's metabolism the productsof the cell's metabolism have to be removed from the reactor or from theculture medium in the reactor. This can be carried out for example byperfusion or dialysis. In the case of dialysis one differentiatesbetween external or internal dialysis.

EP 1 474 223 reports a dynamic mixer. An animal cell culturing devicecomprising a container for the uptake of a cell suspension, a device forfeeding- and discharge of gas into the cell suspension, and a device forproduction of a current in the cell suspension is reported in DE 10 2005053 333. Method and apparatus for cell culture is reported in EP 0 224800. Nomura, T., et al., J. Chem. Eng. Jap. 29 (1996) 134-138 reportdevelopment and mixing characteristics of folding anchor impeller forround bottom flask. An impeller draft tube agitation system forgas-liquid mixing in a stirred tank reactor is reported in WO 01/41919.In U.S. Pat. No. 4,438,074 a continuous polymerization reactor isreported. An agitation system and method for gas transfer into liquidsis reported in EP-A 0 200 886. In EP-B 0 049 229 a cooking apparatushaving a stirring device is reported.

SUMMARY OF THE INVENTION

The agitator as reported herein at least i) allows for rapid mixing ofculture media compared to other stirrers e.g. for introducing correctionagents such as acids or bases via the liquid or cultivation mediumsurface, ii) reduces foam formation, iii) reduces biofouling in dialysisprocesses, and iv) increase mass transfer in dialysis processes due tothe direct orthogonal flow against the dialysis module as a result ofthe combination of differently conveying elements.

Herein is reported an agitator (combination stirrer) comprising at leastone axially-conveying element and at least one radially-conveyingelement relative to the shaft of the agitator wherein the largestdiameter of the at least one axially-conveying element is equal to orless than the inner diameter d_(i) of the radially-conveying element.

One aspect as reported herein is an agitator, comprising

-   -   one radially-conveying element comprising at least two stirrer        blades, and    -   one or more axially-conveying elements each comprising at least        two stirrer blades,    -   wherein the stirrer blades of the radially-conveying element are        parallel to each other and to the shaft axis of the agitator,        and    -   wherein the outer diameter of all axially-conveying elements is        equal to or less than the inner diameter of the        radially-conveying element, and    -   wherein all axially-conveying elements are individually        connected to the radially-conveying element, and    -   wherein all axially-conveying elements are located within the        radially-conveying element, and    -   wherein all conveying elements have a fixed spatial orientation        relative to each other and to the shaft axis of the agitator.

In one embodiment the agitator comprises 1 to 5 axially-conveyingelements, or in another embodiment 1 to 3 axially-conveying elements, orin also an embodiment 1 or 2 axially-conveying elements. In oneembodiment the at least one axially-conveying element is situated in theupper four fifths of the agitator determined from the head of theagitator and is at a maximum distance of h_(4/5) to the head of theagitator. In also an embodiment one axially-conveying element is locatedat a maximum distance of 80% from the top of the blades of theradially-conveying element (i.e. at a maximum distance of 0.8 h) and/orone axially-conveying element is located at a maximum distance of 20%from the top of the blades of the radially-conveying element (i.e. at amaximum distance of 0.2 h). In a further embodiment the at least oneaxially-conveying element and the at least one radially-conveyingelement form together a single element. In another embodiment opposingstirrer blades of the radially-conveying element are linked to eachother by two opposite stirrer blades of an axially-conveying element. Inone embodiment the diameter of all axially-conveying elements isidentical. In another embodiment the axially-conveying elements isselected independently of each other from a propeller agitator,pitched-blade agitator, or inclined-blade agitator.

If the diameter of the axially-conveying element is less than innerdiameter of the radially-conveying element the spatial distance betweenthe tip of the stirrer blade of the axially-conveying element and theinner edge of the radially-conveying element is bridged by a connector.The connector is in one embodiment selected from wire, rod, sheet plateand disc.

In a further embodiment all conveying elements, i.e. theaxially-conveying as well as the radially-conveying elements, rotatewith the same number of rotations per time unit around the shaft axis ofthe agitator when the agitator is operated in a cultivation vessel. Inone embodiment the conveying elements are permanently joined togetherand the agitator consists of one part, i.e. both elements are driven bythe same rotary shaft and have the same number of rotations per timeunit around the shaft axis of the agitator.

The ratio d/D of agitator diameter (d) to cultivation vessel diameter(D) is in one embodiment of from 0.2 to 0.8, in another embodiment offrom 0.3 to 0.6, and in also an embodiment of from 0.33 to 0.5. Inanother embodiment the ratio hid of blade height (h) to agitatordiameter (d) is of from 0.5 to 5, in another embodiment of from 1 to 4,and in also an embodiment of from 1 to 3. In yet another embodiment theratio b/d of agitator blade width (b) to agitator diameter (d) is offrom 0.05 to 0.3, in another embodiment of form 0.1 to 0.25. The term“of from . . . to” denotes a range including the listed boundary values.In another embodiment the stirrer diameter (d) is selected from 500 mm,600 mm, 700 mm, 800 mm, 1000 mm, 1200 mm, 1400 mm, 1600 mm, 1800 mm and2000 mm. In a further embodiment the agitator blade width (b) isselected from, 42 mm, 60 mm, 89 mm, 108 mm, 133 mm.

In one embodiment the one radially-conveying element is an anchorimpeller. In a further embodiment the elements connecting the individualstirrer blades of the radially-conveying element are the stirrer bladesof the axially-conveying element (connecting stirrer blades). In anembodiment the connecting stirrer blades act of from 20% to 100% as anaxially-conveying element, in another embodiment of from 50% to 100% asan axially-conveying element, and in also an embodiment of approximately100% as an axially-conveying element. The term “approximately” denotesthat the given value is the center point of a range spanning plus/minus10% around the value. If this value is a percentage value then“approximately” denotes also means plus/minus 10%, but the value 100%cannot be exceeded.

In a further embodiment the axially-conveying element is aninclined-blade stirrer. In another embodiment the radially-conveyingelement is an anchor impeller and the axially-conveying element is aninclined-blade stirrer where the inclined-blade stirrer is designed toact as a connecting element of blades of the anchor impeller. In anotherembodiment all opposing blades of the radially-conveying element areconnected to each other via blades of an axially-conveying element. Inone embodiment the agitators consists of a combination of one or twoaxially-conveying elements and one radially-conveying element relativeto the shaft axis of the agitator. In another embodiment theradially-conveying element is an anchor impeller and theaxially-conveying elements are two inclined-blade stirrers whereinblades of the inclined-blade stirrers are designed to act as connectingelements of opposing anchor impeller blades.

The ratio h_(SB)/b of the height of the axially-conveying element andthe width of the blades of the radially-conveying element is of from 0.5to 4, in another embodiment of from 0.8 to 3, and also in an embodimentof from 1 to 2. In another embodiment the pitch of the stirrer blades ofthe inclined-blade stirrer is of from 10° to 80°, in a furtherembodiment of from 24° to 60°, and in also an embodiment of from 40° to50° relative to the shaft axis of the agitator.

In one embodiment the radially-conveying element has of from 1 to 8stirrer blades, in another embodiment of from 1 to 4 stirrer blades, andalso in an embodiment 4 stirrer blades. The axially-conveying elementhas in one embodiment of from 1 to 10 stirrer blades, in anotherembodiment of from 2 to 6 stirrer blades, and also in an embodiment 4stirrer blades. In another embodiment the radially-conveying andaxially-conveying elements have the same number of stirrer blades.

In one embodiment the locking device on the shaft of the agitator is inthe lower third seen from the head of the stirrer and has a ratioh_(u)/h_(m) of the height of the reduction part (h_(u)) and the heightof the tightening lid (h_(m)) of from 0.05 to 1, in another embodimentof from 0.2 to 0.8, and in also an embodiment of from 0.3 to 0.5.

In one embodiment the agitator has a height of at least 200 mm, inanother embodiment of from 200 mm to 5000 mm. In another embodiment theheight of the agitator is the height h of the blades of theradially-conveying element.

Herein is further reported as an aspect a device comprising the agitatoras reported herein and a cultivation vessel. In one embodiment theagitator and the cultivation vessel form a functional unit, i.e. theagitator is within the cultivation vessel and can rotate within thecultivation vessel without any spatial limitation. In one embodiment thedevice further comprises a dialysis module. In one embodiment thecultivation vessel is a stirred tank reactor. In a further embodimentthe cultivation vessel is an aerated or submerse gassed stirred tankreactor. In one embodiment the cultivation vessel comprises of from 2 to4 baffles. In another embodiment the baffles are spaced equidistallyaround the circumference of the inside surface of the cultivationvessel.

The ratio d/D of agitator diameter (d) to cultivation vessel diameter(D) is in one embodiment of from 0.2 to 0.8, in another embodiment offrom 0.3 to 0.6, and in also an embodiment of from 0.33 to 0.5. Inanother embodiment the ratio H/D of filling height of the reactionvessel (H) to cultivation vessel diameter (D) is of from 1.0 to 2.5, ina further embodiment of from 1.1 to 2.0, and also in an embodiment offrom 1.4 to 1.8. In one embodiment the cultivation vessel has a workingvolume of from 5 l to 25,000 l.

Herein is also reported as an aspect the use of the agitator or thedevice as reported herein. In one embodiment the use is for thecultivation of cells for the production of a polypeptide, i.e. for thecultivating of cells expressing a polypeptide encoded by a heterologousnucleic acid. In one embodiment the cultivating is a dialysis. In afurther embodiment the cultivating is carried out in a submersed gassedstirred tank reactor. In another embodiment the cell is a eukaryoticcell. In also an embodiment the cell is a mammalian cell. In yet afurther embodiment the cell is selected from a CHO cell, a BHK cell, anNSO cell, a COS cell, a PER.C6 cell, a Sp2/0 cell, and a HEK 293 cell.In one embodiment the polypeptide is an antibody. In a furtherembodiment the antibody is an antibody against CD20, CD22, HLA-DR, CD33,CD52, EGFR, G250, GD3, HER2, PSMA, CD56, VEGF, VEGF2, CEA, Lewis Yantigen, the IL-6 receptor or the IGF-I receptor.

Herein is further reported as an aspect a method for the production of apolypeptide comprising

-   -   a) providing a cell comprising a nucleic acid encoding the        polypeptide,    -   b) providing a device as reported herein,    -   c) cultivating the cell in the device in a cultivation medium        wherein the agitator provides a turbulent flow within the        cultivation vessel, and    -   d) recovering the polypeptide from the cells or the cultivation        medium and thereby producing a polypeptide.

In one embodiment the method is for the cultivating of a cell expressinga polypeptide encoded by a heterologous nucleic acid. In one embodimentthe cultivating is a dialysis. In a further embodiment the cultivatingis carried out in a submersed gassed stirred tank reactor. In anotherembodiment the cell is a eukaryotic cell. In also an embodiment the cellis a mammalian cell. In yet a further embodiment the cell is selectedfrom a CHO cell, a BHK cell, an ISO cell, a COS cell, a PER.C6 cell, aSp2/0 cell, and a HEK 293 cell. In one embodiment the polypeptide is anantibody. In another embodiment the antibody is an antibody againstCD20, CD22, HLA-DR, CD33, CD52, EGFR, G250, GD3, HER2, PSMA, CD56, VEGF,VEGF2, CEA, Lewis Y antigen, the IL-6 receptor or the IGF-1 receptor.

DETAILED DESCRIPTION OF THE INVENTION

Herein reported is an agitator, which is a combination stirrer,comprising a radially-conveying element with one or more defined andintegrated axially-conveying elements. The term “element” denotes a(functional) unit of stirrer blades which are in a fixed spatialconfiguration relative to one another with regard to distance and angle.A radially-conveying element denotes a stirrer which stirrer blades haveno pitch with respect to the shaft axis. An axially-conveying elementdenotes a stirrer which stirrer blades have a pitch with respect to theshaft axis. The stirrer blades of the conveying elements are in oneembodiment rectangular plates although other geometric forms can beused. The conveying direction of an element is denoted with respect tothe shaft axis of the agitator. The axially-conveying elements,especially the individual blades thereof, are arranged as connectingpieces between the blades of the radially-conveying element. Alsoreported herein is a device comprising the agitator and a cultivationvessel. Further reported is the use of the agitator and the device inthe cultivating of cells, in particular in a semi-continuous processsuch as e.g. internal or external dialysis. Exemplary embodiments of theagitators as reported herein are shown in FIG. 1.

Each of the conveying elements consists of a defined number of stirrerblades. Each blade is either directly connected to the rotary shaft oris connected to the rotary shaft via a hub. Each stirrer bladeindependent of the conveying element has an outer edge and an inneredge. The part of each stirrer blade that has the maximum distance tothe shaft axis is denoted as tip of the blade. Each conveying elementhas an outer diameter and an inner diameter. For example, the outerdiameter of an axially-conveying element is the maximum distance betweenthe tips of opposing stirrer blades and the inner diameter of aradially-conveying element is the minimum distance between inner edgesof opposing stirrer blades. In the agitator as reported herein theaxially-conveying element(s) is(are) located within theradially-conveying element. Therefore, the outer diameter of the (all)axially-conveying element(s) is (are) equal to or less than the innerdiameter of the radially-conveying element allowing theaxially-conveying element(s) to be placed inside of theradially-conveying element. Some stirrer blades of (each of) theaxially-conveying element(s) connect opposing stirrer blades of theradially-conveying element. As the radially-conveying elements are notdirectly connected to each other by this connection (each of) theaxially-conveying element is individually, i.e. by itself, connected tothe radially-conveying element. The connecting stirrer blades of theaxially-conveying element do not need to be formed as stirrer blades forthe entire distance between the stirrer blades of the radially-conveyingelement. Thus, the connecting blades of the axially-conveying element donot need to have the function of an axially-conveying over the entiredistance, i.e. the do not have to be formed as a stirrer blade over theentire distance. That is the stirrer blade of the axially-conveyingelement may be shorter than the spatial distance to the inner edge ofthe stirrer blade of the radially-conveying element. This spatial gab isfilled by a connecter that has no special form but provides for theconnection of the tip of the blade of the axially-conveying element tothe inner edge of the blade of the radially-conveying element.

The height h of the radially-conveying element is more than 10 times theheight h_(SB) of the axially-conveying elements. Thus, each of theaxially-conveying elements can be placed at different positions relativeto the height of the radially-conveying element. In one embodiment anaxially-conveying element is located, i.e. it is placed, within amaximum distance of 80% of the total height h of the radially-conveyingelement relative to the head K of the agitator. This denotes that theaxially-conveying element has a maximum distance from the head of theagitator of 0.8 h.

The rotary shall (also termed drive shall) extends through thelongitudinal axis of the cultivation vessel in which the agitator isused.

The modification with respect to a primarily radially-conveyingimpeller, e.g. to an anchor impeller, is the (axially)downwards-conveying inclined-blade stirrer which is integrated in theupper and/or middle and/or lower region of the radially-conveyingelement. The agitator as reported herein provides among other things foran improved mixing of liquid solutions, for example in order tointroduce correcting agents such as e.g. an acid or a base, nutrientsolutions, anti-foaming agents or also CO.sub.2 or O.sub.2 via theliquid surface. Furthermore, the agitator reduces foam formation and ina dialysis process reduces biofouling and increases mass transfer by adirect orthogonal flow against the dialysis module. It has been foundthat the additional axially-conveying element(s) surprisingly does(do)not increase shear stress while at the same time improving the mixingand mass transport in the dialysis module.

In semi-continuous culturing processes, such as e.g. external orinternal dialysis, substrates are fed to the reactor across a membraneand at the same time inhibiting components/metabolic products of thecultured cells are lead away. This exchange of material is by diffusion.The main influence factors therefore are the prevailing concentrationdifference, the membrane material, the membrane surface, the diffusioncoefficients of the respective compounds inside the membrane materialand the thickness of the phase interface which is determined by the flowagainst the membrane.

When dialysis is employed in high cell density fermentation it is aperfusion-like, semi-continuous process in which a (hollow fiber)dialysis module attached in the reactor provides the exchange areabetween the culture medium and fresh nutrient medium. The nutrientmedium is pumped from a storage container through the dialysis moduleand thereafter returned again into the storage container (for aschematic diagram see FIG. 2). The dialysis module can be locatedoutside the reactor (external dialysis) or within the reactor (internaldialysis). The same physical laws apply to both operating modes.

Thus, reported herein is a device comprising an agitator as reportedherein and a cultivation vessel. In one embodiment the device alsocomprises a dialysis module. The components of the device aredimensioned in a way that they can exert their intended function, i.e.the cultivation vessel can take up the cultivation medium, the agitatorcan mix the medium and disperse added compound therein and the dialysismodule can provide fresh medium and lead away metabolic compoundssecreted by the cultivated cell. Thus, the agitator has a diameter thatallows for an unhampered rotation within the vessel in the presence andabsence of the dialysis module. It has been found that with the deviceas reported herein a high cell density cultivation as perfusioncultivation or as dialysis cultivation can be carried outadvantageously. The cultivating is carried out in one embodiment at arotation speed of the agitator at which a Reynolds-number independentconstant power input to the cultivation medium can be achieved, i.e.during the cultivating a turbulent cultivation medium flow in thecultivation vessel is provided. It is possible with a device as reportedherein to cultivate shear sensitive mammalian cells at a lower rotationspeed of the agitator but at the same power input compared to knownstirrers.

The form of the cultivation vessel is not limited. In one embodiment thecultivation vessel is a cylindrical vessel. In another embodiment thecultivation vessel is a stirred tank reactor. The cultivation vessel mayhave any dimension. In one embodiment the cultivation vessel has aworking volume of from 5 l to 25,000 l.

Components from the fresh nutrient medium diffuse from the interior ofthe dialysis module through the semi-permeable hollow fiber membraneinto the reactor and at the same time metabolites of the cultivatedcells diffuse in the opposite direction from the reactor into thenutrient medium according to the concentration difference. The aim is tokeep the absolute concentration of inhibiting metabolites in the reactoras low as possible (dilution) and at the same time to maintain theconcentration of essential nutrients as long as possible at an optimallevel for the culture. This results in improved culture conditionscompared to a process without dialysis enabling to achieve highermaximum cell density or product titer.

The transport processes in the dialysis module can be described in anequivalent manner to mass transfer on a gas bubble by the two-filmtheory in conjunction with the first Fick's law. Thus, assuming lineargradients based on the exchange area A_(H) of the hollow fiber dialysismodule, the effective transferred diffusion flux J_(eff) is according toequation 1:

$\begin{matrix}{J_{{eff},i} = {{{- D_{{eff},i}} \cdot A_{H} \cdot \frac{c_{2,i} - c_{1,i}}{x_{2} - x_{1}}} = {{- D_{{eff},i}} \cdot A_{H} \cdot {\frac{{\Delta c}_{i}}{z_{eff}}.}}}} & {\left( {{Equation}\mspace{14mu} 1} \right).}\end{matrix}$

The driving force of diffusion is the concentration difference Δ_(Ci)between the inside and outside of the dialysis module relative to theeffective diffusion path z_(eff). This effective diffusion path iscomposed of the individual paths through the inner laminar boundarylayer on the inner side of the hollow fiber membrane of the dialysismodule δ_(B1), through the hollow fiber membrane itself δ_(M) andthrough the outer laminar boundary layer on the outer side of the hollowfiber membrane in the reactor δ_(H1) (see FIG. 3). They generally dependon the size and shape of the diffusing molecule, the properties of thesurrounding medium and the temperature. Separate mass transfercoefficients can be defined for the respective individual sections andfrom the summation of their reciprocals the total mass transferresistance 1/k can be given according to equation 2:

$\begin{matrix}{\frac{1}{k} = {\frac{1}{k_{Hl}} + \frac{1}{k_{M}} + {\frac{1}{k_{Bl}}.}}} & {\left( {{Equation}\mspace{14mu} 2} \right).}\end{matrix}$

The transport resistances in the laminar boundary layers on the innerand outer side of the hollow fiber membrane also depend on the flowagainst the follow fiber membrane. The better, i.e. the moreperpendicular, the flow towards the membrane is the narrower the laminarboundary layers become and the lower are the corresponding transportresistances.

For a dialysis module in a reactor a direct dependency of the transportresistance of the outer laminar boundary layer on among others from thefollowing factors exists:

-   -   the speed of rotation of the stirrer,    -   the type of stirrer,    -   the flow against the membranes,    -   the primary flow profile generated by the stirrer.

The resistance of the laminar boundary layer on the inner side of thehollow fiber membrane can be neglected due to the low inner diameter andthe concomitant high flow velocities. Within this application the term“inner side of the hollow fiber membrane” denotes the side of the hollowfiber membrane which faces the storage container. The term “outer sideof the hollow fiber membrane” denotes the side of the hollow fibermembrane which faces the reactor. The total mass transfer resistance isthus a series resistance to which mainly the resistance within themembrane and the resistance of the outer laminar boundary layercontribute (Rehm, et. al., Biotechnology—volume 3: Bioprocessing, VCHWeinheim, 1993). The total mass transfer coefficient k results from thereciprocal total mass transfer resistance and can be related to thesurface area by multiplication with the volume-specific surface a of thehollow fiber dialysis module (ka value).

The following equation 3 can be used to describe the concentration timecourses for the balance spaces reactor and storage container:

$\begin{matrix}{\frac{d\; c_{R}}{dt} = {{ka} \cdot {\left( {c^{*} - c_{R}} \right).}}} & {\left( {{Equation}\mspace{14mu} 3} \right).}\end{matrix}$

The typical concentration time courses in the reactor c_(R) and thestorage container c_(V) are shown in FIG. 4.

In general submerse gassed reactors are used in cell culture. In thesecases a one-stage or two-stage axially-conveying stirrer system ismainly used. This generates a flow profile which is essentially parallelto the rotary shaft of the employed stirrer. Thus, in the arrangement asshown in FIG. 2 with the dialysis module parallel to the rotary shaft adirect flow against the dialysis module is not achieved. This has adisadvantageous effect on the mass transport in the dialysis module(wider outer laminar boundary layer on the fiber surface).

A direct tangential or radial flow against the dialysis membrane has anadvantageous effect which can for example be achieved by a standardanchor impeller. This impeller generates a flow which is directeddirectly onto the dialysis module or modules in the reactor and thusreduces the laminar boundary layer on the surface of the dialysismodule(s). This simple radial flow is, however, disadvantageous for theother basic technical process functions in particular with regard tomixing the reactor and mass transfer especially in submersed gassedreactors. The gas can be introduced into the cultivation vessel e.g. viaa pipe sparger or a ring sparger.

It has been found that the agitator (combination stirrer) as reportedherein provides for a direct orthogonal flow against or towards thedialysis module and at the same time is suitable for mixing in liquidsat/from the surface of the culture medium and for rapid total mixing ofthe culture medium whereby the shear stress for the cells in the reactoris almost not increased when compared to other stirring systems. It hasturned out that the axial flow generated by the axially-conveyingelement(s), i.e. for example by inclined-blade stirrers, ensures amixing of the culture medium in a short time. In addition shearsensitive cells such as mammalian cells can be cultivated at the sameshear stress with increased mixing efficiency by using the agitator asreported herein.

The agitator as reported herein combines or integrates the properties ofa radially-conveying element, i.e. for example of an anchor impeller,which are important for an application in dialysis processes with thoseof an axially-conveying element, such as e.g. an inclined-blade stirrer,which are important to fulfill the basic technical process requirementsor functions. Exemplary embodiments of the agitator as reported hereinare shown in FIGS. 1a ) to 1 f) in which one or more of the connectingelements of a radially-conveying element (anchor impeller) are designedas axially-conveying element fined-blade stirrer). Specific embodimentsare shown in FIGS. 1a ) to 1 f).

The rotary speed of the stirrer n is used as a characteristic velocityand the stirrer width d is used as a characteristic length.

In fluid dynamics the Reynolds's number describes the ratio of inertialforce to inner frictional force in a hydrodynamic system. Therefrom alsostatements can be made about the degree of turbulence of the movedmedium. For stirred liquids the stirrer Reynolds's number is defined inequation 4 to be:

$\begin{matrix}{{Re} = {\frac{n \cdot d^{2}}{\nu} = {\frac{n \cdot d^{2} \cdot \rho}{\eta}.}}} & \left( {{Equation}\mspace{14mu} 4} \right)\end{matrix}$

The Newton number (also referred to as power number) describes the ratioof resistance force to flow force and is thus a measure for the flowresistance of a stirrer in a stirred material and is described inequation 5:

$\begin{matrix}{{Ne} = {\frac{P}{\rho \cdot n^{3} \cdot d^{5}}.}} & \left( {{Equation}\mspace{14mu} 5} \right)\end{matrix}$

Agitators with a low Newton number, such as propeller or inclined bladestirrers, convert the power input more efficiently in hydrodynamicoutput, i.e. fluid motion, than those with a high Newton number, such asrushton turbines.

A criterion for assessing the stirring processes in culture orcultivation processes is the mixing time. The “mixing time” in aninhomogeneous liquid-liquid mixture denotes the time which is requiredto achieve a defined homogeneity in the culture medium. Factorsinfluencing the mixing time are the degree of mixing and the site ofobservation. The degree of mixing in turn depends on the reactorgeometry, the stirrer geometry, the rotary frequency of the stirrer, andthe substances of the stirred materials.

It is important for culture or cultivation processes that, as far aspossible, all cells are optimally and uniformly supplied with thenecessary substrates (such as nutrient medium, O₂) and that metabolites(such as overflow products, CO₂) are concomitantly led away. This meansthat repositories and sinks that may occur spatially as well astemporarily in the reactor have to be avoided or minimized in order toavoid damage to the cells. This can be achieved e.g. by using an adaptedstirring system for mixing the reactor's contents. The mixing processescan be divided into the sub-processes micro-mixing and macro-mixing.Micro-mixing is defined as the molecular concentration adjustment due todiffusion or microturbulences; in contrast macro-mixing is defined asthe convective coarse mixing caused by the stirrer (see e.g. Houcine,I., et al., Chem. Eng. Technol. 23 (2000) 605-613; Zlokarnik, M.,“Rührtechnik Theoric und Praxis”, Springer Publishers, BerlinHeidelberg, 1999). The degree of mixing can according to Henzler(Henzler, “Homogenisieren: Referenz-Rührsysteme und Methoden zurErfassung der Homogenisierungseigenschaften von Rührsystemen”, GVCFachausschuß Mischvorgänge, 1998) be defined as follows in equation 6:

$\begin{matrix}{\mathcal{X}_{1} = {1 - {\frac{\Delta\; a}{\overset{\_}{a}}.}}} & {\left( {{Equation}\mspace{14mu} 6} \right).}\end{matrix}$

In this equation ā corresponds to the concentration of the tracersubstance after a theoretically complete mixing and Δa corresponds tothe maximum difference between the local concentrations of the tracersubstance at a time t. In general a degree of intermixing of X₁=0.95 isregarded as sufficient (see Henzler, supra). The mixing coefficientC_(H 0.95) as described in equation 7 is based on this degree of mixingand is the product of the mixing time θ_(0.95) and the rotary speed ofthe stirrer n that was used. Thus, it corresponds to the number ofstirrer revolutions which are required after adding a correcting agentin order to achieve a degree of intermixing of 0.95 and results fromequation 7:C _(H 0.95)=Θ_(0.95) ·n  (Equation 7).

The mixing coefficients as shown in Table 1 and in extracts in FIG. 5were determined by using the decolorizing method and result from themixing time investigations (mixing characteristic see equation 7). Amaximum relative error of ±15% was determined by multiple measurements.

TABLE 1 Mixing coefficients. Stirrer H/D d/D C_(H)(Re) agitator asreported herein (KR) 1.6 0.4 31 stirrer with a standard disk stirrer(1SSR) 1.0 0.33 42 stirrer with three separate inclined- 1.6 0.33 64blade stirrers (3SBR) stirrer with three separate standard 1.6 0.33 77disk stirrers (3SSR)

From the data presented in Table 1 it can be seen that the agitator asreported herein (KR) provides for a considerably shorter average mixingindex of approximately 31 at a constant Reynolds's number for example incomparison to a stirrer combination consisting of three separateinclined-blade stirrers (3SBR) with an average mixing coefficient of 64.In the experiments it can be seen that the sodium thiosulfate is rapidlysucked into the liquid near the shaft of the agitator.

Another process-relevant parameter for the culture or cultivation ofanimal cells is the shear stress applied to the cell in the culturemedium. Animal cell cultures are, among others, limited by themechanical and hydrodynamic stress applied to the cells. The stress is,on the one hand, caused by the stirrer itself and, on the other hand, bythe bubble aeration of the culture medium (see e.g. Wollny, S., andSperling, R., Chem. Ing. Tec. 79 (2007) 199-208). For the turbulent flowregions which are mostly predominant in stirred tank reactors, thehydrodynamic stress is given by the Reynolds's stress approach accordingto equation 8 (Henzler, H. J. and Biedermann, A., Chem. Ing. Tec., 68(1996) 1546-1561):τ_(turb) =ρ·u′ ².  (Equation 8).

According to equation 8 the main stress can be deduced as being due tothe turbulent velocity fluctuation u′ of the fluid elements.

The characterization of shear stress was carried out in non-gassed statewith an incorporated dialysis module. The reference particle diametersd_(VF) that were determined are shown in FIG. 6.

It can be seen that with the agitator as reported herein one of thebiggest reference particle diameters can be obtained and thus this isthe system with the lowest shear at a constant power input. Thus, at thesame shear forces for example for the cultured cells it is possible toachieve a higher power input which, due to a higher number ofrevolutions, leads to higher turbulences and a higher flow against thedialysis module as well as to a better complete mixing.

In addition to NaCl, glucose was also used as a tracer substance toexperimentally determine mass transfer coefficients. In general it couldbe shown that the mass transfer of the dialysis module is, on the onehand, influenced by the power input and, on the other hand, by thestirrer geometry, i.e. the primary generated flow mode. Tangential orradial flow profiles have proven to be particularly suitable with regardto reducing the outer laminar boundary layers around the hollow fiberdialysis membranes (higher mass transfer rates). The highest masstransfer coefficients can be achieved in each case by the agitator asreported herein.

FIG. 7 shows a comparison of the Newton numbers. As can be seen theagitator as reported herein provides for a considerably higher Newtonnumber. At the same time it can be seen that the power number isindependent of the Reynolds number.

Thus, the agitator provides for an increased power number independent ofthe Reynolds number when operated to produce a turbulent flow within thereaction vessel.

FIG. 8 shows that the highest mass transfer coefficients of the dialysismodule at a constant volume-specific power input can be achieved withthe agitator as reported herein. This is due to the radial flowgenerated by the stirrer according to the invention which, in comparisonto the standard stirrer systems, allows a better flow against the hollowfiber dialysis module.

Compared to standard stirrer systems the agitator as reported hereinprovides for considerable advantages in the mixing (FIG. 5, Table 1),the generated shear stress (FIG. 6), as well as in the mass transfercoefficients in dialysis (FIG. 8). It is particularly remarkable that,despite the introduced modifications, no significantly higher shearstresses can be measured (see e.g. Pohlscheidt, M., et. al., Chem. Ing.Tec. 80 (2008) 821-830).

The abbreviations used in this application have the following meanings(see also FIG. 1a ):

-   -   b: width of the blades of the radially-conveying element    -   d: agitator total outer diameter    -   d_(w): diameter of the shaft    -   h: height of the stirrer blades of the radially-conveying        element    -   h_(m): height of the fastening sleeve    -   h_(SB): height of an axially-conveying element    -   h_(u): height of the reducer    -   Δh: height difference of two axially-conveying elements    -   l: length of the stirrer blades of an axially-conveying element    -   α: blade pitch of the blades of an axially-conveying element    -   z: number of stirrer blades per stirrer    -   d_(i): inner distance between the stirrer blades of the        radially-conveying element    -   h_(4/5): 4/5 height from above of h    -   K: stirrer head, i.e. the highest point of the stirrer when it        is not attached to a rotary shaft    -   D: cultivation vessel inner diameter    -   H: filling height of the cultivation vessel.

In one embodiment the ratio of the height difference (Δh) of twoaxially-conveying elements to the cultivation vessel diameter (D) is atleast 0.75.

Herein is reported the use of the agitator as reported herein for theculture or cultivation of cells for the recombinant production ofproteins or antibodies as an aspect. In one embodiment the culture is adialysis. In a further embodiment the culture is carried out in asubmersed gassed stirred tank reactor. In another embodiment the cell isa eukaryotic cell, in another embodiment a mammalian cell. In yet afurther embodiment the cell is a CHO cell, a BHK cell, an NSO cell, aCOS cell, a PER.C6 cell, a Sp2/0 cell or a HEK 293 cell. In oneembodiment the cell is selected from Arthrobacter protophormiae,Aspergillus niger, Aspergillus oryzae, Bacillus amyloliquefaciens,Bacillus subtilis, BHK cells, Candida boidinii, Cellulomonas cellulans,Corynebacterium lilium, Corynebacterium glutamicum, CHO cells, E. coli,Geobacillus stearothermophilus, H. polymorpha, HEK cells, HeLa cells,Lactobacillus delbruekii, Leuconostoc mesenteroides, Micrococcus luteus,MDCK cells, Paenebacillus macerans, P. pastoris, Pseudomonas species, S.cerevisiae, Rhodobacter species, Rhodococcus erythropolis, Streptomycesspecies, Streptomyces anulatus, Streptomyces hygroscopicus, Sf-9 cells,and Xantomonas campestris. In yet a further embodiment the antibody isan antibody against CD20, CD22, HLA-DR, CD33, CD52, EGFR, G250, GD3,HER2, PSMA, CD56, VEGF, VEGF2, CEA, Lewis Y antigen, IL-6 receptor orIGF-1 receptor.

The following example and figures are provided to illustrate the subjectmatter of the invention. The protective scope is defined by the attachedpatent claims. It is clear that modifications can be made on the subjectmatter of the disclosed methods without departing from the subjectmatter of the invention.

DESCRIPTION OF THE FIGURES

FIG. 1 Schematic diagram of various embodiments of the combinationstirrer according to the invention; b: width of the stirrer blade; d:stirrer diameter; d_(w): diameter of the rotary shaft; h: height of thestirrer blade of the radially-conveying stirrer; h_(m): height of thefastening sleeve; h_(SB): height of the axially-conveying stirrer;h_(u): height of the reducer; l: length of the stirrer blade of theaxially-conveying stirrer; α: blade pitch of the axially-conveyingstirrer; z: number of stirrer blades per stirrer; d_(i): inner distancebetween the stirrer blades of the radially-conveying stirrer; h_(4/5):4/5 height from above of h; K: stirrer head; a) general scheme of theagitator as reported herein; b) to f) embodiments of the agitator asreported herein; g) scheme of a radially conveying element seen from thetop along the shaft axis; h) scheme of an axially conveying element seenfrom the top along the shaft axis; i) scheme of an embodiment of theagitator as reported herein seen from the top along the shaft axisshowing the connection of opposing stirrer blades of theradially-conveying element via connecting stirrer blades of theaxially-conveying element; j) scheme of an embodiment of the agitator asreported herein seen from the top along the shaft axis showing theconnection of opposing stirrer blades of the radially-conveying elementvia connecting stirrer blades of the axially-conveying element whereinthe diameter of the axially-conveying element is less than the innerdiameter of the radially-conveying element and the spatial distance isbridged by a connector.

FIG. 2 Schematic diagram of a device for dialysis cultivation.

FIG. 3 Schematic diagram of the concentration gradients on the hollowfibers of the dialysis module.

FIG. 4 Typical concentration time course in the reactor (C_(R)) andstorage container (C_(V)).

FIG. 5 Comparison of the mixing coefficients C_(H) for different stirreras a function of the Reynolds's number (Re); KR=combination stirrer asreported herein; SSR=standard disk stirrer; SBR=inclined-blade stirrer.

FIG. 6 Reference flake diameter d_(VF) as a function of thevolume-specific power input and stirrer configuration; KR=combinationstirrer as reported herein; SSR=standard disk stirrer;SBR=inclined-blade stirrer; Pohlscheidt, M., et al.=Chem. Ing. Tec. 80(2008) 821-830.

FIG. 7 Diagram of the power coefficient Ne of different stirrer as afunction of the Reynolds's number (Re); KR=combination stirrer asreported herein; SSR=standard disk stirrer; SBR=inclined-blade stirrer.

FIG. 8 Mass transfer coefficient k_(a) in the dialysis as a function ofthe specific power input; KR=combination stirrer as reported herein;SBR=inclined-blade stirrer.

EXAMPLE 1

Cultivation Vessel

All investigations were carried out in a 100 l Plexiglas® modelcontainer (referred to as DN 440 in the following).

EXAMPLE 2

Power Input

The power input of different stirrer was determined by measuring thetorque on the rotary shaft. A data processing system model GMV2 togetherwith the torque sensor model DRFL-II-5-A (both from the “ETHMesstechnik” Company, Gschwend, Germany) were used to record the torque.For each stirrer system the torque was firstly recorded at variousrevolution speeds in the unfilled state (M_(empty)) and subsequently bymeans of a triplicate determination in the filled state (M_(load))according to equation 9:M=M _(load) −M _(empty).  (Equation 9).

Afterwards the corresponding Newton number (Ne number) and theReynolds's number (Re number) were calculated for each point. Since theNewton numbers for a stirrer system become constant in the turbulentflow region, the calculated Newton numbers were subsequently averaged inthis region (Uhl, V. W. and Gray, J. B., Mixing Theory and Practice,Academic Press, 1966). This mean represents the total Newton number ofthe respective stirrer.

EXAMPLE 3

Homogenization

The homogenization was determined using the color change method as wellas using the conductivity method.

The color change method is based on the decolorization of a starchsolution stained with iodine-potassium iodide by addition of sodiumthiosulfate (I, KI, starch, Na₂S₂O₃ obtained from the Carl Roth GmbH &Co KG Company, Karlsruhe, Germany). A one molar sodium thiosulfatesolution and a one molar iodine-potassium iodide solution (Lugol'ssolution) as well as a starch solution at a concentration of 10 g/l wereused as the starting solutions. In correspondence with the conductivityexperiments, at least four speed steps were examined per stirrer(quadruplicate determinations per speed step) in which a maximum of fourexperiments were carried out per container filling amount. In each casethe starch solution was added once per filling of the container. Foreach individual measurement the corresponding volume of theiodine-potassium iodide solution was firstly added and subsequently thesodium thiosulfate was added. The mixing time has been determinedmanually from the time point at which the sodium thiosulfate was addedand one second was subtracted in each case in order to take the additiontime into consideration. After completion of the measurement thecontainer filling volume was titrated (neutralized) withiodine-potassium iodide in order to compensate for the excess of thepreviously added sodium thiosulfate.

In the conductivity method the mixing time is defined as the time fromaddition of an electrolyte solution to the time at which the measuredconductivity fluctuations for the last time exceed a tolerance range of±5% around the conductivity values which are reached in a stationarystate. If several probes are used, the longest detected mixing time ineach case is regarded as representative for the entire system.

A 30% (w/v) NaCl solution (NaCl crystalline, Merck KGaA Company,Darmstadt, Germany) was used as an electrolyte solution to determine themixing time by the conductivity method. This was added in pulses ontothe liquid surface at the rotary shaft of the stirrer and the volume peraddition was selected such that the jumps in conductivity which resultedin a stationary state did not exceed 200 mS/cm.

For each stirrer at least four speed steps were examined. The mixingtime was determined at least eight times per speed step and these eightvalues were averaged. The mixing coefficient of the respective stirrersystems is given as the mean of the mixing coefficients averaged perspeed step. The conductivity was in each case measured by three 4-poleconductivity probes (TetraCon, WTW Company, Weilheim) at various radialor axial positions in the container. The conductivity signals were readout online via the measuring amplifier that was used (Cond813, Knick“Elektronische Messgeräte GmbH & Co, KG” Company, Berlin, Germany). Themeasured values were stored online and simultaneously for all probes bymeans of the software Paraly SW 109 (Knick “Elektronische MessgeräteGmbH & Co, KG” Company, Berlin, Germany) at a sampling rate of 5seconds. After the series of measurements was completed the data wereevaluated separately for each probe.

EXAMPLE 4

Shear Stress

A model particle system, the blue clay polymer flake system, was used todetermine shear stress. This is a model particle system consisting of acationic polymer (Praestol BC 650) and a clay mineral (blue clay) whichis placed in the vessel. A flocculation reaction is started by addingPraestol BC 650 which generates flakes of a defined size. These flakesare subsequently broken up by the mechanical and hydrodynamic stress ofthe stirrer system. In the case of bubble-gassed systems they areadditionally broken up by the energy dissipation when the bubbles areformed and burst. The average particle diameter of the model particlesystem was used as a measured variable to characterize the shear stress.In this case the change in the particle size was measured in situ by aFocused Beam Reflectance Measurement Probe from the Mettler ToledoCompany (referred to as FBRM® in the following). The rates of change inparticle size that were determined are a measure for the shear stressprevailing in the model system. The gradient of the rate of change ofparticle size becomes smaller during the course of the experiment but noequilibrium state is formed (particle comminution down to a diameter ofthe blue clay primary particles of ≈15 μm). For this reason an end flakediameter d_(P50)′ for the blue clay polymer flake system was determinedaccording to the following criterion (equation 10):

$\begin{matrix}{\left. {\frac{d\left( d_{P50} \right)}{dt} \leq {0.0055\left\lbrack {\mu\;{m/s}} \right\rbrack}}\rightarrow d_{P50} \right. = {d_{P50}^{\prime}.}} & {\left( {{Equation}\mspace{14mu} 10} \right).}\end{matrix}$

In order to ensure comparability of the end flake diameters at differentpower inputs and between different stirrers, the reference flakediameter was calculated as follows (equations 11 to 13):

$\begin{matrix}{d_{VF} = {{m \cdot d_{P\; 50}^{\prime}} - {b.}}} & \left( {{Equation}\mspace{14mu} 11} \right) \\{m = {{1.3 \cdot 10^{- 6} \cdot \left( \frac{P}{V} \right)^{2}} + {1.37 \cdot 10^{- 3} \cdot \left( \frac{P}{V} \right)} + {2.46.}}} & \left( {{Equation}\mspace{14mu} 12} \right) \\{b = {{8.12 \cdot 10^{- 5} \cdot \left( \frac{P}{V} \right)^{2}} + {6.48 \cdot 10^{- 3} \cdot \left( \frac{P}{V} \right)} + {76.9.}}} & {\left( {{Equation}\mspace{14mu} 13} \right).}\end{matrix}$

TABLE 2 Substances used to determine the particle stress (concentrationsare based on the container fill volume). Ingredient ConcentrationManufacturer Wittschlicker 5 g/l Braun Tonbergbau Co., Germany blue clayPraestol 650 BC 5 ml/l Stockhaus GmbH & Co. KG, (solution 2 g/l)Krefeld, Germany NaCl 1 g/l Merck KGaA, Darmstadt, Germany CaCl₂ — CarlRoth GmbH & Co. KG, (solution 30 g/l) Karlsruhe, Germany

Firstly the 100 l model container was filled with a corresponding volume(H/D ratio) of completely demineralized water (VE water) and maintainedat a temperature of 20° C. Subsequently the conductivity was adjusted toa value of 1000 μS/cm by titration with a CaCl₂ solution. Theconductivity was measured by a 4-pole conductivity probe (probe:TetraCon, WTW Co. Weilheim; measuring amplifier: Cond813, Knick“Elektronische Messgeräe GmbH & Co, KG” Company, Berlin, Germany).Afterwards the blue clay and the NaCl were added in appropriate amountsto the solution. Subsequently a homogenization phase took place at thehighest speed with a duration of at least 20 minutes. The FBRM® probe(FBRM® Lasentec® D600L, Mettler-Toledo GmbH Co., Giessen, Germany) wasmounted in the container perpendicular from above (immersion depth 300mm) at a radial distance of 70 mm to the wall. The flocculation reactionwas subsequently started by adding Praestol 650 BS at a defined speed.The measured values were recorded online by means of the program dataacquisition control interface version 6.7.0 (Mettler-Toledo GmbH,Giessen, Germany). The reference flake diameter was determined from themeasurement data. At least three power inputs were measured for eachstirrer. In each case three measurements were carried out per powerinput.

EXAMPLE 5

Dialysis (Mass Transfer Liquid—Liquid)

A NaCl solution (NaCl crystalline, Merck KGaA Company, Darmstadt,Germany) was used as a tracer substance to determine the concentrationhalf-life of the module (DIADYN-DP 070 F1 OL; MICRODYN-NADIR GmbHCompany, Wiesbaden, Germany) in relation to the stirrer system that wasused and the volume-specific power input. The tracer substance wasadjusted in the storage container at the start of each experimental runto a base-line conductivity of 1500 μS/cm. The reactor was filled withcompletely demineralized water for each experimental run. The fillingvolume of the reactor was 100 l (H/D=1.6) and that of the storagecontainer was 400 l (H/D=2.0) and both containers were maintained at atemperature of 20° C. at the start of each experiment. The conductivityin both containers was measured by a 4-pole conductivity probe (probe:TetraCon, WTW Co. Weilheim, Germany; measuring amplifier: Cond813, Knick“Elektronische Messgeräte GmbH & Co, KG” Company, Berlin, Germany). Thesampling rate of the measurement amplifiers that were used was 5 secondsand the measurement values were stored online and simultaneously for allprobes by means of the software Paraly SW 109 (Knick “ElektronischeMessgeräte GmbH & Co, KG” Company, Berlin, Germany). The NaCl solutionwas circulated by means of a peristaltic pump between supply containerand dialysis module (housing pump 520 U, Watson-Marlow GmbH, Company,Rommerskirchen, Germany) at a constant flow rate of 2.1 For theevaluation, probe 1 was used as a reference probe for the reactor andprobe 3 was used as a reference probe for the storage container. Thedata of these two probes were evaluated by an evaluation routine. Ineach case at least six different power inputs in the reactor wereinvestigated per stirrer.

In order to compare the mass transfer characteristics determined bymeans of the NaCl solution, additional measurements were carried outwith a glucose solution as tracer substance. The experimental setup wasnot changed for this. A defined glucose concentration (glucose solid,Merck KGaA. Company, Darmstadt, Germany) at a concentration of 3 g/l wasprovided in the storage container. The glucose concentration wasdetermined manually and simultaneously for the storage container andreactor at a time interval of 10 minutes by means of a blood sugarmeasuring instrument (ACCU-CHEK® Aviva, Roche Diagnostics GmbH Company,Mannheim, Germany).

The invention claimed is:
 1. An agitator configured to improve mixingand mass transport within a cell cultivation vessel, comprising: a shaftcomprising a shaft axis; one anchor impeller comprising at least twoopposing stirrer blades, wherein the stirrer blades of the anchorimpeller have no pitch with respect to the shaft axis; and one or moreinclined-blade stirrers each comprising at least two stirrer blades,wherein the stirrer blades of each of the inclined-blade stirrers have apitch with respect to the shaft axis, wherein the stirrer blades of theanchor impeller are parallel to each other and to the shaft axis,wherein an outer diameter of each inclined-blade stirrer is equal to orless than an inner diameter of the anchor impeller, the outer diameterof each inclined-blade stirrer is a maximum distance between tips ofopposing stirrer blades of each inclined-blade stirrer and the innerdiameter of the anchor impeller is a minimum distance between inneredges of opposing stirrer blades of the anchor impeller, wherein allinclined-blade stirrers are individually connected to the anchorimpeller, wherein all inclined-blade stirrers are located within theanchor impeller, and wherein the anchor impeller and the inclined-bladestirrers all have a fixed spatial orientation relative to each otherwith regard to distance and angle.
 2. The agitator according to claim 1,wherein the number of inclined-blade stirrers is 1 or 2 or
 3. 3. Theagitator according to claim 1, wherein one of the inclined-bladestirrers is located at a maximum distance of 80% from a top of theblades of the anchor impeller and/or one of the inclined-blade stirrersis located at a maximum distance of 20% from a top of the blades of theanchor impeller.
 4. The agitator according to claim 1, wherein theopposing stirrer blades of the anchor impeller are linked to each otherby two opposite stirrer blades of one or more of the inclined-bladestirrers.
 5. The agitator according to claim 1, wherein the pitch of thestirrer blades of the inclined-blade stirrers is between 10° and 80°relative to the shaft axis of the agitator.
 6. The agitator according toclaim 1, wherein the anchor impeller has up to 8 stirrer blades.
 7. Theagitator according to claim 1, wherein the inclined-blade stirrers haveindependent of each other up to 10 stirrer blades.
 8. The agitatoraccording to claim 1, wherein the anchor impeller has a height of atleast 200 mm.